Fredericks, A. C. | Search bioRxiv

bioRxiv2025

Mosquito NF-κB-mediated innate immunity exerts arbovirus-specific antiviral effects at multiple stages of the viral life cycle

One third of all emerging infectious diseases are vector-borne, with the vectors ecology and physiology playing key roles in determining whether viruses can access new vertebrate host species and spread globally. Innate immunity is a known barrier to virus replication in mosquito vectors that influences arboviral vector tropism. We here generated novel CRISPR-Cas9-mediated knockouts of the NF-{kappa}B family transcription factor Rel2 in Aedes aegypti-derived Aag2 cells and tested the impact on the replication of a diverse range of arboviruses in the Flaviviridae and Togaviridae families and the class Bunyaviricetes. We found that NF-{kappa}B-mediated innate immunity has broad antiviral activity against the Ae. aegypti-borne orthoflaviviruses dengue virus (DENV), yellow fever virus (YFV) and Zika virus (ZIKV) in mosquito cells. In contrast, little impact of NF-{kappa}B-loss-of-function was observed for the alphavirus chikungunya virus (CHIKV) or phlebovirus Rift Valley fever virus (RVFV), indicating specificity in the antiviral effects of NF-{kappa}B-mediated immunity. By comparing orthoflaviviruses with different transmission routes (mosquito-borne, tick-borne, no known vector), we demonstrated that NF-{kappa}B-mediated immunity exerts its antiviral effects both early and late in the viral replication cycle, and that NF-{kappa}B-mediated immunity is not the only molecular barrier influencing the ability of orthoflaviviruses to replicate in Ae. aegypti cells. Overall, our work demonstrates the importance of mosquito NF-{kappa}B-mediated innate immunity in suppressing arbovirus replication, and shows that the barriers for arboviruses to adapt to new vector species are multifactorial and virus-specific. Our findings increase our understanding of the molecular barriers influencing arboviral emergence, and could inform the development of refractory mosquitoes incapable of transmitting human pathogens. Author SummaryMosquito-borne viruses are causing an increasing global disease burden due to urbanisation, globalisation, changing land use and the climate change-driven invasion of mosquitoes into new locations. Few vaccines and no medicines for mosquito-borne viral infections are licensed for human use. Historically, the most effective way to reduce human disease has been to kill mosquitoes using insecticides, which also harms beneficial insects and is leading to resistance. Newer technologies with lower ecological impacts are being developed, including genetically modified mosquitoes with a reduced ability to transmit viruses. Like humans, mosquitoes have an immune system that protects against viral infections, and strengthening this immune system through genetic modification has shown promise for reducing virus transmission. We here expand our understanding of the ways in which mosquito immunity could be harnessed by demonstrating that a gene called "Rel2" helps the mosquito fight mosquito-borne virus infection. Unlike previously studied mosquito immune responses, Rel2 has a very broad (but not universal) protective capacity against viruses. Our work also indicates that Rel2 works in concert with other arms of the mosquito immune system, highlighting a need for further research to fully realise the potential of genetically modifying mosquito immune responses for the prevention of human disease.

microbiology↗

bioRxiv2019

Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the impact of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

BackgroundAedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections that more closely mimic the situation in nature has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures.\n\nMethodology/Principle FindingsWe generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies.\n\nConclusions/SignificanceOur findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.\n\nAUTHOR SUMMARYMosquito-borne viruses usually only infect humans through the bite of a mosquito that carries the virus. Viruses transmitted by the yellow fever mosquito Aedes aegypti, including dengue virus, Zika virus, yellow fever virus and chikungunya virus, are causing an ever-increasing number of human disease cases globally. Mosquito-borne viruses have to infect and replicate inside the mosquito before they are transmitted to humans, and the presence of other infectious agents can change the efficiency of virus transmission. Mosquitoes are known to be infected with insect-specific viruses that only infect mosquitoes and cannot cause human disease. We have shown here that in laboratory cell cultures derived from the Aedes aegypti mosquito, pre-existing infection with an insect-specific virus called Phasi Charoen-like virus does not affect the infection and growth of the mosquito-borne viruses dengue virus, Zika virus, Sindbis virus or vesicular stomatitis virus. Compared to previous research, our research is more reflective of conditions that mosquito-borne viruses encounter in nature, and our results provide important new insights into whether and how insect-specific viruses affect mosquito-borne virus transmission. Ultimately, this information could inform ongoing research into whether insect-specific viruses could be used to prevent the transmission of mosquito-borne viruses to reduce global disease burdens.

microbiology↗